| Poplar is an important timber forest and shelter forest tree species in China,but in recent years it has suffered from the threat of pests and diseases,among which canker of Lonsdalea quercina is a bacterial disease that causes serious harm to poplar.Gene expression of poplar infected with Lonsdalea quercina subsp.Populi has been studied,but the mechanism causing the differential expression of these genes has not been elucidated.Recent studies have shown that epigenetic regulation is one of the important regulatory mechanisms for plants to cope with biological stress,and DNA methylation is an important part of epigenetics.The analysis of the changes of DNA methylation after infection,the correlation of methylation with different resistance among poplars,and the function of disease-related miRNA will help to reveal and improve the regulatory mechanism of poplar disease resistance and provide reference for the application of epigenetic regulation in genetic improvement of tree disease resistance.In this study,poplar 107 and Populus tomentosa ‘Henan’ were inoculated with L.quercina,to study the methylation changes of poplar after inoculation and the differences of DNA methylation among different resistant materials.In addition,in order to analyze the dynamic changes of DNA methylation in susceptible poplar,DNA methylation analysis was performed at different days of post ioculation(2 d,4 d and 6 d).In combination with the previous transcriptome and miRNA researches of our group,the relationship between the methylation differences of the corresponding DNA and disease resistance was further analyzed.In previous research of our group,miR1447 and miR6427 were differentially expressed significantly and multiple target genes were probably associated with adversity stress.These two miRNAs were transformed into poplar,to clarify their resistance regulation functions.The results of this investigation are given as follows:(1)The total cytosine methylation ratio of the two poplar materials decreased after inoculation,and the methylation ratio of the Populus tomentosa was lower than that of poplar 107 both uninoculation and after inoculation.The methylation ratio of CHH in the resistant material P.tomentosa ‘Henan’ was also decreased after inoculation,and the methylation ratio of CHH in the resistant material was lower than that of the susceptible material both uninoculation and after inoculation.(2)With the change of pathogen infection time,the proportion of total cytosine methylation of poplar 107 in the pathogen infection of 2 d,4 d and 6 d generally showed a gradually decreasing trend.The change trend of CHH methylation was consistent with the general change trend of methylation,which was consistent with previous reports that CHH methylation was inhibited by pathogens.(3)The sites of these DNA methylation differences were further annotated to various parts of the gene,it was found that the promoter and repeat regions of a large number of genes in poplar 107 and P.tomentosa ‘Henan ’ were demethylated at 6 d after being infected by the pathogen.The main difference is that the quantity of promoter region and repeat region of poplar107 with CHH methylation is about 2 times of that in P.tomentosa.Combined with the existing transcriptome data,a gene annotated to calcium-dependent protein kinase was up-regulated and the CHH methylation was down-regulated in the promoter region,which implied that this gene might be involved in the regulation of resistance of poplar to pathogen.(4)Pathogen infection reduced the number of miRNAs with CHH methylattion in the 5’ Flanking region of P.tomentosa ‘Henan’ and 107,and the number of miRNAs with methylation in 107.In addition,when the combined analysis of DNA methylation and expression was performed on 13 miRNAs with few methylation types and locations,no significant correlation was found between changes in expression level and CG/CHG/CHH methylation and its occurrence area,but 12 of the 13 miRNAs were involved in changes in CHH methylation.(5)After the inoculation of L.quercina,the expression levels of miR1447 and miR6427 of two poplars were down-regulated and up-regulated respectively.DNA methylation of miR1447 and miR6427 was down-regulated in poplar 107 of the affected material,while that of miR1447 and miR6427 was up-regulated or unchanged in P.tomentosa ‘Henan’ of the disease-resistant material.Moreover,the expression levels of miR6427 and miR1447 target genes were negatively correlated with miR6427 and miR1447,suggesting that DNA methylation might occur simultaneously on miRNAs and their target genes,which imply that the changes of DNA methylation in the genomes of miR6427 and miR1447 are relatively complex,but the changes in the DNA methylation of miR1447 and miR6427 in the two materials.are related to the changes in CHH methylation.(6)The transgenic poplar seedlings overexpressing miR1447 or miR6427 and the transgenic plants with inhibited expression of miR1447 were successfully obtained by leaf disc transformation,which provided a solid basis for the subsequent studies on the functions of these two miRNAs in poplar disease resistance.This investigation should provide a new theoretical clue to the elucidation of the mechanism of different resistance among poplar varieties and initial basis for the methylation mediated regulation of resistance in trees.The results would give the reference to the further uncover of miRNAs regulation mechanism of disease resistance and the application of miRNAs in molecular improvement of tree resistance. |
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